Methenyltetrahydrofolate synthetase (MTHFS) expression enhances folate-dependent de novo purine biosynthesis. In this study, the effect of increased MTHFS expression on the efficacy of the glycinamide ribonucleotide formyltransferase (GARFT) inhibitor LY309887 was investigated in SH-SY5Y neuroblastoma. GARFT catalyzes the incorporation of formate, in the form of 10-formyltetrahydrofolate, into the C8 position of the purine ring during de novo purine biosynthesis. SH-SY5Y neuroblastoma with increased MTHFS expression displayed a 4-fold resistance to the GARFT inhibitor LY309887, but did not exhibit resistance to the thymidylate synthase inhibitor Pemetrexed. This finding supports a mechanism whereby MTHFS increases the availability of 10-formyltetrahydrofolate for GARFT. MTHFS expression is elevated in animal tumor tissues compared to surrounding normal tissue, consistent with the dependence of transformed cells on de novo purine biosynthesis. The level of MTHFS expression in tumors may predict the efficacy of antipurine agents that target GARFT.